Attempts to make sense of the antiphospholipid syndrome.

Many investigators have been intrigued by the paradoxical association of a circulating anticoagulant, first called lupus anticoagulant by Feinstein and Rapaport [1], with a tendency to develop thrombosis, as initially described by Walter Bowie [2]. Work in Leuven on this topic started when Luis Carreras, an Argentinian hematologist, joined the laboratory of blood coagulation at this university in 1979. At that time, the head of the laboratory was Marc Verstraete. Luis had a particular interest in antibody-mediated coagulation disorders, and had prepared reviews on thrombosis and thrombocytopenia induced by heparin [3] and on the lupus inhibitor [4]. In Leuven, he joined Jos Vermylen, senior member of the laboratory, and an internist with particular interest in hemostasis, thrombosis and vascular disease. As such, Professor Vermylen was involved in both laboratory research and patient care.

Link between anti-CD36 antibodies and thrombosis in the antiphospholipid syndrome.

OBJECTIVE: Some studies have previously suggested the involvement of antibodies directed against CD36 (anti-CD36) in the pathogenesis of thrombosis. The aim of this study was to evaluate the prevalence of anti-CD36 in patients with antiphospholipid antibodies (aPL) and its relationship with thrombosis. METHODS: Anti-CD36 were tested using an indirect MAIPA assay in 62 patients with autoimmune aPL but without SLE; there were 38 with and 24 without thrombosis. Nineteen patients with thrombosis served as an aPL(-) control group and 58 healthy subjects as the normal control group. RESULTS: 15 of 62 aPL patients (24.2%) but only 1 of 58 (1.7%) normal controls had anti-CD36 (p < 0.0005). As compared to normal controls, the prevalence of anti-CD36 was significantly higher in aPL patients with (26.3%, p < 0.0005) or without thrombosis (20.8%, p < 0.01). Anti-CD36 were significantly more frequent in aPL patients with thrombosis than in thrombosis aPL(-) subjects (26.3% vs 0%, p = 0.02). The presence of anti-CD36 seems to be more frequent in aPL patients with recurrent thrombosis than in those with a single episode (36.8% vs 15.8%). CONCLUSION: The presence of anti-CD36 is highly prevalent in patients with autoimmune aPL with a trend to being more frequent in patients with recurrent episodes of thrombosis.

Markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with antiphospholipid antibodies.

OBJECTIVE: To evaluate plasma levels of markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with or without antiphospholipid antibodies (aPL) and to compare them to those found in patients with antiphospholipid syndrome (APS). METHODS: 42 patients with leprosy (35 lepromatous and 7 borderline): 29 aPL(+) and 13 aPL(-), as well as 26 healthy subjects as normal controls (NC) and 79 control aPL patients without leprosy (59 with and 20 without APS) were included in the study. Plasma soluble P and E selectin (sPsel and sEsel), and VCAM-1 (sVCAM-1), prothrombin F1 + 2 fragment (F1 + 2), thrombin-antithrombin complexes (TAT) and D dimer (DD) were measured by ELISA. The protein C pathway was assessed by the ProC global test. RESULTS: Leprosy patients with aPL presented increased median levels of sPsel [ng/ml (82.0 vs 36.0, p < 0.001)] and sVCAM-1 [ng/ml (495 vs 335, p < 0.001)] compared to NC, as observed in control aPL patients without leprosy. Levels of sPsel in aPL(+) patients with leprosy were significantly higher than in aPL(-) ones (52.5 ng/ml), p = 0.005. However, plasma markers of thrombin generation were increased in control aPL patients without leprosy but not in those with leprosy. ProcC global test was abnormal in 24.1% of leprosy patients with aPL compared to 4.4% of NC (p < 0.024), and to 57.2% of control patients with aPL without leprosy (p = 0.005). CONCLUSIONS: We demonstrated that although patients with leprosy present a high prevalence of aPL, and platelet and endothelial cell activation in vivo to the same extent than patients with APS, they do not show a procoagulant state.

Markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with antiphospholipid antibodies

OBJECTIVE: To evaluate plasma levels of markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with or without antiphospholipid antibodies (aPL) and to compare them to those found in patients with antiphospholipid syndrome (APS). METHODS: 42 patients with leprosy (35 lepromatous and 7 borderline): 29 aPL(+) and 13 aPL(-), as well as 26 healthy subjects as normal controls (NC) and 79 control aPL patients without leprosy (59 with and 20 without APS) were included in the study. Plasma soluble P and E selectin (sPsel and sEsel), and VCAM-1 (sVCAM-1), prothrombin F1 + 2 fragment (F1 + 2), thrombin-antithrombin complexes (TAT) and D dimer (DD) were measured by ELISA. The protein C pathway was assessed by the ProC global test. RESULTS: Leprosy patients with aPL presented increased median levels of sPsel [ng/ml (82.0 vs 36.0, p smaller 0.001)] and sVCAM-1 [ng/ml (495 vs 335, p smaller 0.001)] compared to NC, as observed in control aPL patients without leprosy. Levels of sPsel in aPL(+) patients with leprosy were significantly higher than in aPL(-) ones (52.5 ng/ml), p = 0.005. However, plasma markers of thrombin generation were increased in control aPL patients without leprosy but not in those with leprosy. ProcC global test was abnormal in 24.1 per cent of leprosy patients with aPL compared to 4.4 per cent of NC (p smaller 0.024), and to 57.2 per cent of control patients with aPL without leprosy (p = 0.005). CONCLUSIONS: We demonstrated that although patients with leprosy present a high prevalence of aPL, and platelet and endothelial cell activation in vivo to the same extent than patients with APS, they do not show a procoagulant state.

Major and potential prothrombotic genotypes in a cohort of patients with venous thromboembolism.

Factor V Leiden (FVL) and the prothrombin 20210A (PT-20210A) variant are well-known risk factors for venous thromboembolism (VT). The thermolabile variant (TT) of the methylenetetrahydrofolate reductase (MTHFR) gene, and homozygosity for the 4G allele of the promoter region of the plasminogen activator inhibitor-1 (PAI-1) are potential genetic polymorphisms that have not been consistently associated with increased risk of VT. A case-control study was performed on 192 consecutive unrelated patients referred for evaluation of thrombophilia because of VT and 200 healthy controls. FVL was found in 10.4% of patients compared to 3.0% of controls, while 6.3% of patients were carriers of the PT-20210A allele compared to 2.0% of controls. The adjusted odds ratios (OR) were 5.92 and 4.03 for FVL (P=.001) and the PT-20210A (P=.033), respectively. The prevalence of homozygotes for MTHFR (TT) and PAI-1 (4G/4G) among patients and controls were 13.7% versus 13.0% and 21.6% versus 23.5%, respectively (P=ns). A total of 121 patients underwent a complete screening for FVL, the PT-20210A, protein C (PC), protein S (PS), antithrombin III (ATIII), levels of factor VIII, and antiphospholipid antibodies (aPL). In 59 patients (48.8%) at least one defect was found, being a single defect in 55 and combined defects in 4 patients. Plasma levels of homocysteine (Hcy) were measured in 138 patients and 144 controls. Subjects from both groups carrying the MTHFR-TT variant had higher Hcy levels than those with the normal genotype. Hyperhomocysteinemia (HHcy) by itself is a risk factor for VT (OR 4.92, P<.0001). We conclude that FVL and the PT-20210A are risk factors for VT as well as Hcy levels, but the MTHFR and PAI-1 polymorphisms do not appear to be associated with VT in our country.

Thrombophilic factors in chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTE-PH) is an infrequent cause of pulmonary hypertension that develops in 0.1-0.2% of patients who survive after an acute venous thromboembolic event. According to the largest series so far reported, 15-30% of patients with diagnosis of CTE-PH have an underlying congenital or acquired hypercoagulable state. To determine the prevalence of thrombophilic factors in our population, we analyzed 24 patients admitted to our institution between November 1992 and March 2000 fulfilling criteria for CTE-PH. Eighteen patients disclosed abnormal results in the screening for thrombophilia. The presence of antiphospholipid antibodies (lupus anticoagulant and/or anticardiolipin antibodies) was the abnormality most frequently found (12 out of 24 patients). We found hyperhomocysteinaemia in 7/14, true protein S deficiency in 1/10, protein C deficiency in 1/13, activated protein C resistance in 1/22, antithrombin III deficiency in 1/24, and prothrombin gene G20210A mutation in 1/18 patients. Factor V Leiden was normal in all 18 patients studied. Five patients (20.8%) disclosed more than one thrombophilic abnormality. In conclusion, contrary to the largest series of patients with CTE-PH so far reported, we found that 75% of patients with CTE-PH presented at least one thrombophilic risk factor, being antiphospholipid antibodies in 50% of the cases. We recommend a thorough screening for thrombophilia in all patients with diagnosis of CTE-PH.

Increased lipid peroxidation correlates with platelet activation but not with markers of endothelial cell and blood coagulation activation in patients with antiphospholipid antibodies.

Recent studies have shown that patients with antiphospholipid antibodies (aPL) have increased lipid peroxidation. We evaluated the urinary excretion of 11-dehydro thromboxane B2 (11-DH-TXB(2) and isoprostane F(2alpha)III (IPF(2alpha)III), reflecting platelet activation and lipid peroxidation in vivo, and plasma soluble markers of endothelial cell, platelet and blood coagulation activation: soluble vascular cell adhesion molecule-1 (sVCAM-1), P- and E-selectin (sPsel and sEsel), F1 + 2 fragment of prothrombin (F1 + 2), thrombin-antithrombin complexes (TAT) and D-Dimer (DD). We studied 79 patients with aPL (47 with previous thrombosis), 45 healthy volunteers (normal controls, NC), 12 patients with systemic lupus erythematosus (SLE) without aPL and a thrombosis control group (TCG) without thrombophilia (n = 16). Urinary levels (mean, range) of eicosanoids and isoeicosanoids were significantly increased in 39 patients with aPL compared with 25 NC, 11-DH-TXB(2) 164.0 ng/mmol creatinine (9.5-1162.8) versus 43.4 ng/mmol creatinine (4.2-87.6), P < 0.001; IPF(2alpha)III 56.9 pg/mg creatinine (5.5-388.7) versus 27.0 pg/mg creatinine (4.6-87.6), P = 0.03. Both metabolites were significantly correlated (rho = 0.49, P = 0.014), but none correlated with any clinical manifestation or antibody profile. The aPL group presented increased levels of sPsel, sEsel, sVCAM-1, TAT, F1 + 2 and DD, but any soluble marker correlated with IPF2alphaIII. Urinary 11-DH-TXB(2) correlated with sPsel (rho = 0.39, P = 0.04). Compared with SLE controls, the SLE group with aPL had higher levels of F1 + 2. Plasma levels of F1 + 2 and DD were significantly increased and a trend to higher sPsel was found in aPL patients with thrombosis compared with the TCG. Platelet activation, lipid peroxidation and blood coagulation activation seem to be important in the pathophysiology of antiphospholipid syndrome.

The combination of thrombophilic genotypes is associated with definite antiphospholipid syndrome.

BACKGROUND AND OBJECTIVES: Thrombosis and pregnancy morbidity are clinical features of the definite antiphospholipid syndrome (APS). These clinical complications are also associated with the presence of inherited thrombophilias. Interactions between acquired and genetic risk factors are becoming increasingly related to a higher thrombotic risk. The aim of our study was to determine the prevalence of four common gene polymorphisms in patients with antiphospholipid antibodies (aPL). DESIGN AND METHODS: A series of 105 consecutive unselected patients with aPL grouped as having APS (n= 69) and not having APS (n= 36) was studied. A control group of 200 healthy subjects was also investigated for the presence of factor V Leiden (FVL), the 20210A allele of the prothrombin (PT-20210A) gene, the thermolabile variant (677TT) of methylenetetrahydrofolate reductase (MTHFR), and the 4G/4G genotype of the plasminogen activator inhibitor (PAI-1) promoter. RESULTS: Two patients who belong to the APS group carried the FVL while PT-20210A was found in 6 patients with APS (8.7%) and in 1 of the non-APS group (2.8%). The prevalence of FVL was similar to that found in the control group whereas PT-20210A was significantly more frequent in APS patients than in normal controls (2.0%, p=0.02). The MTHFR-677TT was found in 22.0%, 15.1% and 13.0%, and the PAI-1 (4G/4G) in 27.5%, 22.8% and 23.5% of APS, non-APS patients and normal controls, respectively. Furthermore, combinations of PT-20210A or FVL with PAI-1 (4G/4G) were significantly more frequent in APS patients (5.8%) than in normal controls (0.5%, p=0.016). This difference was not found between non-APS patients and normal subjects. INTERPRETATION AND CONCLUSIONS: Present data indicate that testing for heritable thrombophilia would be important to identify aPL subjects with an increased risk of developing APS.

High prevalence of antiphospholipid antibodies in leprosy: evaluation of antigen reactivity.

Antiphospholipid antibodies (aPL) have been reported not only in autoimmune disorders but also in various infectious diseases. Accumulating evidence indicates that beta2 glycoprotein I (beta2GPI) and prothrombin are the main proteins to which autoimmune aPL bind. The aim of this study was to evaluate the prevalence of different aPL in patients with leprosy. We included 51 outpatients (42 lepromatous and 9 borderline leprosy) without any clinical feature of the antiphospholipid syndrome (APS). 35 had lupus anticoagulant and 31 had anticardiolipin antibodies (aCL). Anti-beta2GPI antibodies were highly positive in 29/51 and anti- prothrombin antibodies (anti-II) were detected in 23/51. Almost all aCL and anti-beta2GPI were of IgM isotype, while IgG isotype was more frequent among anti-II. No statistical difference was found when aPL were evaluated in patients grouped according to their bacteriological status. Furthermore, patients under treatment (n=33) had a similar frequency of positive aPL compared to patients in vigilance (n=14). Assessing the specificity of antibody binding to CL and beta2GPI in ELISA by means of inhibition studies with cardiolipin-beta2GPI liposomes, leprosy and APS sera showed a similar behaviour. Comparable results were also found in both groups of patients when inhibition experiments with lysate of Mycobacterium leprae were carried out. In summary, leprosy-related aPL resemble those found in patients with APS but the immunoglobulin isotype is different, with IgM much more prevalent in leprosy patients.

Which are the best biological markers of the antiphospholipid syndrome?

The diagnosis of antiphospholipid syndrome (APS) requires the presence of both clinical and biological features. Due to the heterogeneity of anti-phospholipid antibodies (aPL) the laboratory approach for their detection includes clotting-based tests for lupus anticoagulant (LA) as well as solid-phase assays for anticardiolipin antibodies (aCL). In addition, as it has been shown that autoimmune aPL recognize epitopes on phospholipid (PL)-binding plasma proteins, assays detecting antibodies to beta 2-glycoprotein I (beta 2-GPI) or prothrombin have been developed. The association between venous or arterial thrombosis and recurrent fetal loss with the presence of conventional aPL (LA and/or aCL) has been confirmed by many studies. The LA and IgG aCL at moderate/high titre seem to exhibit the strongest association with clinical manifestations of the APS. Several reports indicate that LA is less sensitive but more specific than aCL for the APS. Assays against PLs other than CL as well as the use of mixtures of PLs have been proposed to improve the detection of APS-related aPL. Concerning antibodies to PL-binding proteins (detected in the absence of PLs), there is evidence that anti-beta 2-GPI are closely associated with thrombosis and other clinical features of the APS. Moreover, these antibodies may be more specific in the recognition of the APS and in some cases may be present in the absence of aPL detected by standard tests. Many issues are still under debate and are discussed in this review, such as the problems of standardization of anti-beta 2-GPI assays, detection of the IgA isotype of aCL and anti-beta 2-GPI, the coagulation profiles of LA in the recognition of the thrombotic risk and the association of particular markers with subsets of patients with APS.

Occurrence of anti-prothrombin and anti-beta2-glycoprotein I antibodies in patients with history of thrombosis.

New evidence indicates that antibodies to beta2-glycoprotein I (anti-beta2GPI) or to human prothrombin (anti-II)(or to both of these) are specific markers of the antiphospholipid syndrome (APS). They have been mainly associated with thrombotic complications in patients with APS. However, some studies have reported that elevated levels of anti-II, but not of anfi-beta2GPI, imply a risk of venous thrombosis (VT) or arterial thrombosis (AT) in subjects with no previous thrombosis and no antiphospholipid antibodies (aPL) by ELISA. The present study Included 180 patients with a history of thrombosis, 83 of them without aPL (group I) and the remaining 97 diagnosed as having APS (group II). Anti-beta2GPI was found in only 1 of the 83 patients from group I but was found in approximately 50% of those from group II (P < .0001). In contrast, positive anti-II was detected with a high prevalence in patients from group I (VT, 22.6%; AT, 26.7%) and in those from group II (VT, 37.5%; AT, 14.6%). No statistical differences were found in the prevalence of anti-II between the two groups of patients. On the other hand, such a difference was significant when compared with results in a normal group (1/67, 1.4%, P < .0001). These data Indicate that anti-II occurs frequently in patients with previous thrombosis either with or without lupus anticoagulant activity. Accordingly, testing of anti-II might be clinically useful in the evaluation for thrombophilla.

Binding properties of antibodies to prothrombin and beta2-glycoprotein I (beta2-GPI) assayed by ELISA and dot blot.

Most anti-phospholipid antibodies (aPL) associated with the anti-phospholipid syndrome are autoantibodies with specificity towards beta2-GPI (anti-beta2-GPI) or prothrombin (anti-II). They are mainly screened by ELISA using polyoxygenated plates. However, some authors have claimed that immunoblotting can also be used. Exposure of cryptic epitopes or increase of antigen density on its binding to either phospholipids or suitable plastic surfaces are the two hypotheses proposed for the interaction of beta2-GPI or prothrombin with their antibodies. Forty-five patients with aPL were studied: 25 with lupus anti-coagulant (LA) and anti-cardiolipin antibodies (aCL), 10 with LA alone and 10 with aCL but negative LA. All patients with LA and aCL were positive for anti-beta2-GPI by ELISA and dot blot, while 15/25 had anti-IIELISA and 14 of them also had anti-II by dot blot assay. No patient with LA alone tested positive for anti-beta2-GPI by ELISA or dot blot, whereas 6/10 had anti-IIELISA (five of them were also positive by dot blot). Four out of 10 aCL-positive patients had anti-beta2-GPI by ELISA and dot blot, while none of this group had anti-II by ELISA or dot blot. Antibody binding to beta2-GPI or prothrombin in both ELISA and dot blot was significantly reduced by phospholipid liposomes mixed together with beta2-GPI or prothrombin, whereas liposomal eluants retained it in both assays. Parallel fluid-phase inhibition experiments using increasing concentrations (up to 200 microg/ml) of beta2-GPI or prothrombin demonstrated that antibody binding reduction was more evident on dot blot than on ELISA. It was almost completely abolished on dot blot, while on ELISA a moderate inhibition was achieved even at the highest protein concentration. However, antibody binding on ELISA was virtually abolished when diluted sera were incubated with high protein concentrations applied to nitrocellulose membranes. We could infer that ELISA and dot blot detect antibodies with some differences in avidity but directed against native epitopes on beta2-GPI and prothrombin.

Different types of antiphospholipid antibodies in AIDS: a comparison with syphilis and the antiphospholipid syndrome.

Alloimmune antiphospholipid antibodies react with phospholipids and are an epiphenomenon of an infectious disease. Most autoimmune antiphospholipid antibodies recognise phospholipid-protein complexes or proteins, such as beta2 glycoprotein I or prothrombin and are related to the clinical features of the antiphospholipid syndrome. Lupus anticoagulant, anticardiolipin antibodies, antiprothrombin, and anti-beta2 glycoprotein I antibodies were studied in 61 human immunodeficiency virus (HIV) patients, 55 syphilis patients, and 45 selected patients with antiphospholipid syndrome. Lupus anticoagulant was present in 72% of HIV and 81% of antiphospholipid syndrome patients. None of the syphilis patients had lupus anticoagulant. Anticardiolipin antibodies were found at comparable prevalence in the three groups (HIV 67%, syphilis 67%, antiphospholipid syndrome 84%). HIV had more frequently anti-beta2 glycoprotein I (13%) and antiprothrombin (12%) antibodies than syphilis (0 and 4%, respectively), but significantly less than antiphospholipid syndrome (61 and 40%, respectively). Autoimmune antiphospholipid antibodies in HIV without clinical features of antiphospholipid syndrome might be a reflex of the immunological chaos and/or the constant antigenic virus stimulus.

Antiplatelet factor 4--heparin antibodies in patients with antiphospholipid antibodies.

Antibodies directed against platelet factor 4-heparin are present in patients with heparin-induced thrombocytopenia (HIT). Additionally, it has been suggested that heparin can be an antigenic target of antiphospholipid antibodies (aPL). We investigated the presence of heparin-platelet factor 4-induced antibodies (HPIA) in 33 patients with aPL. There were 30 patients with lupus anticoagulant, 25 with anticardiolipin antibodies, 21 with anti-beta2 glycoprotein I, and 18 with antiprothrombin antibodies. 20 patients had a history of thrombosis and 19 had received heparin during the last 60 months. We found 7 (21.2%) who had HPIA; 5 of them also had anti-beta2 glycoprotein I antibodies. Four patients had severe thrombocytopenia and suspicion of HIT. Among them, two presented high positive HPIA results, one of them with positive platelet aggregation test. The third patient showed grey zone HPIA and borderline aggregation test and the fourth one had negative results. Among patients without a history of HIT, 2 who had never received heparin presented high positive, one a moderate positive, and one a grey zone HPIA result; all of them with negative aggregation tests. Five positive sera samples were incubated with cardiolipin liposomes in the presence of beta2 glycoprotein I, and whereas an inhibition greater than 50% was achieved in anticardiolipin and anti-beta2 glycoprotein I activities, HPIA results did not change. We demonstrate that HPIA could be frequently found in patients with aPL. They are responsible for HIT in some cases but can also be found in patients who have not received heparin. Whether they predispose patients with aPL to HIT is not known; nevertheless, a close follow-up of heparin treatment in these patients seems to be mandatory.

Early occlusion of coronary by-pass associated with the presence of factor V Leiden and the prothrombin 20210A allele: case report.

The presence of factor V Leiden mutation and the variation of the prothrombin gene 20210GA have been described as additional risk factors for arterial thrombosis when other acquired or metabolic risk factors are present. We report here a 56-year-old man who developed coronary artery disease since 1980 without any known risk factor and underwent a cardiopulmonary by-pass in 1997. In the first month after surgery, he became symptomatic, and an angiography showed complete occlusion of the grafts and some native coronary arteries. Three months after the second cardiopulmonary by-pass, a thrombophilic state was searched, and plasma levels of lipoprotein (a) (LPa) were measured. The patient is heterozygous for factor V Leiden mutation and has the variation 20210GA of the prothrombin gene and high levels of LPa. These findings induced us to add oral anticoagulation to the aspirin treatment, and the patient is in a good condition 11 months later.